US9187570B2 - Fusion protein - Google Patents

Fusion protein Download PDF

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US9187570B2
US9187570B2 US13/923,974 US201313923974A US9187570B2 US 9187570 B2 US9187570 B2 US 9187570B2 US 201313923974 A US201313923974 A US 201313923974A US 9187570 B2 US9187570 B2 US 9187570B2
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binding
peptide portion
peptide
dps
portion capable
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US20140045247A1 (en
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Ippei Inoue
Ichiro Yamashita
Bin Zheng
Hisashi Yasueda
Yukiharu Uraoka
Yasuaki Ishikawa
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Ajinomoto Co Inc
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Ajinomoto Co Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a fusion protein. Specifically, the present invention relates to a fusion protein capable of forming a multimer having an internal cavity, a multimer of the fusion protein, and a complex comprising the multimer of the fusion protein, and the like.
  • Ferritin forms a 24-meric structure, and stores iron that is a metal essential for living organisms in an internal cavity formed by its structure.
  • Ferritin-like proteins are ubiquitously present in organisms from animals and plants to microorganisms, and profoundly involved in homeostasis of an iron element in the living organisms and cells.
  • Dps DNA-binding protein from starved cells.
  • Dps forms a 12-meric structure consisting of a monomer unit having a molecular weight of about 18 kDa, thereby forming a cage-like structure having an external diameter of 9 nm which has an internal cavity with a diameter of about 5 nm, and can store an iron molecule as an iron oxide nanoparticle in this internal cavity.
  • ferritin is able to artificially store the nanoparticles including oxides of metals such as beryllium, gallium, manganese, phosphorus, uranium, lead, cobalt, nickel, and chromium, and semiconductors/magnetic substances such as cadmium selenide, zinc sulfide, iron sulfide and cadmium sulfide, in addition to iron.
  • metals such as beryllium, gallium, manganese, phosphorus, uranium, lead, cobalt, nickel, and chromium
  • semiconductors/magnetic substances such as cadmium selenide, zinc sulfide, iron sulfide and cadmium sulfide, in addition to iron.
  • peptides capable of binding to the inorganic material or the organic material are developed by screening using a phage for the purpose of making a complex of a biomaterial and an inorganic material or an organic material.
  • a phage for the purpose of making a complex of a biomaterial and an inorganic material or an organic material.
  • the peptides that recognize carbon nanotube (CNT) and carbon nanohorn (CNH) Non-patent Literature 2, Patent Literatures 1 and 3
  • titanium oxide Patent Literature 2
  • gold Non-patent Literature 3
  • zinc oxide Non-patent Literature 5
  • germanium oxide Non-patent Literature 6
  • zinc sulfide and cadmium sulfide Non-patent Literature 7 and the like are known as such peptides.
  • Nanographite structures such as CNT and CNH that are carbon crystalline structures are expected to be applied to electronic materials, catalysis, optical materials, medical technology, and the like by constructing a complex with the other nanomaterial based on their electric properties and structures.
  • Technology of constructing a nanocomplex by attaching the nanographite structure with metal nanoparticles using ferritin fused with a CNH binding peptide is reported (Non-patent Literature 2 and Patent Literature 3).
  • Titanium oxide generates an electron having a reduction capacity and a hole having an oxidation capacity on its surface by light energy when receiving the light.
  • titanium oxide is attempted to be applied to antimicrobial materials, deodorant materials, air cleaning materials, anti-stain materials, hydrogen generating catalysts, solar cells, and the like (Non-patent Literature 11).
  • hydroxide ion is oxidized utilizing the oxidation capacity generated on the surface of titanium oxide by the light, radical having strong oxidation capacity can be generated.
  • the radical can enhance biocidal effects, effects of decomposing odor substances such as acetaldehyde and ammonia, effects of decomposing harmful substances such as NOx and formaldehyde in air, and effects of decomposing dusts by its oxidation capacity. It is also attempted that water is electrolyzed utilizing the generated oxidation reduction capacities to produce oxygen and hydrogen and the hydrogen is utilized as clean energy. Titanium oxide can be utilized as the solar cell by isolating excited electrons generated in titanium oxide by light. Titanium oxide can be functioned as the solar cell by adsorbing a dye as an enhancer to the surface of the titanium oxide and isolating the excited electrons generated by irradiating the dye with light.
  • the increase of the surface area of the titanium oxide can increase total numbers of electrons having reduction capacity and holes having oxidation capacity which are generated by light energy, and a total number of dyes adsorbed to the surface.
  • the enhancement of the electric properties can decrease a probability that electrons excited by light are bound again to holes. Thus, more electrons and oxidation capacity can be obtained.
  • Patent Literature 4 technology of laminating an oxide film and nanoparticles by arranging the nanoparticles on a silicon substrate, forming a titanium oxide film or a silicon oxide film on the nanoparticles, and arranging the nanoparticles on the oxide film, which technology utilizes a metal encapsulating protein, ferritin fused with titanium oxide. It is also reported that a protein encapsulating metal nanoparticles of cobalt or an iron oxide is oriented on a pattern depicted by titanium utilizing the metal encapsulating protein, ferritin fused with titanium oxide (Non-patent Literature 8).
  • Non-patent Literature 10 a CNT surface is coated with titanium oxide to change the electric property of CNT by using a polypeptide consisting of 35 amino acid residues in which a CNT-binding peptide was fused to a titanium oxide-binding peptide.
  • Non-patent Literature 11 Technology of coating the carbon nanotube with titanium oxide using virus (Non-patent Literature 11) and technology of coating the carbon nanotube with titanium using polyoxometalate (Non-patent Literature 12) is also reported.
  • Patent Literature 1 International Publication No. WO2006/068250.
  • Patent Literature 2 International Publication No. WO2005/010031.
  • Patent Literature 3 JP Publication No. 2004-121154.
  • Patent Literature 4 International Publication No. WO2006/126595.
  • Non-patent Literature 1 I. Yamashita et al., Biochem Biophys. Acta, 2010, vol. 1800, p. 846.
  • Non-patent Literature 2 S. Wang et al., Nat. Mater., 2003, vol. 2, p. 196.
  • Non-patent Literature 3 S. Brown, Nat. Biotechnol., 1997, vol. 15, p. 269.
  • Non-patent Literature 4 R. Tsukamoto et al., WSEAS Trans. Biol. Biomed., 2006, vol. 36, p. 443.
  • Non-patent Literature 5 K. Kjaergaard et al., Appl. Enbiron. Microbiol., 2000, vol. 66, p. 10.
  • Non-patent Literature 6 M. B. Dickerson et al., Chem. Commun., 2004, vol. 15, p. 1776.
  • Non-patent Literature 7 C. E. Flynn et al., J. Mater. Chem., 2003, vol. 13, p. 2414.
  • Non-patent Literature 8 K. Sano et al., Nano Lett., 2007, vol. 7, p. 3200.
  • Non-patent Literature 9 K. Iwahori et al., Chem. Mater., 2007, vol. 19, p. 3105.
  • Non-patent Literature 10 M. J. Pender et al., Nano Lett., 2006, vol. 6, No. 1, p. 44.
  • Non-patent Literature 11 M. A. Fox and M. T. Dulay, Chem. Rev., 1993, vol. 93, p. 341.
  • Non-patent Literature 12 Xiangnan Dang et al., Nature Nanotechnology, 2011, vol. 6, p. 377-384.
  • Non-patent Literature 13 Bin Fei et al., Nanotechnology, 2006, vol. 17, p. 1589-1593.
  • a multimer of a fusion protein comprising a polypeptide portion capable of forming a multimer having an internal cavity, and a first peptide portion capable of binding to a first target substance and a second peptide portion capable of binding to a second target substance can be useful for producing the devices, materials and the like that are excellent in photocatalytic activity, electric properties or the like, and completed the present invention.
  • the present invention is as follows.
  • the fusion protein, the multimer, and the complex of the present invention are useful for the production of the novel devices having the enhanced electric properties and/or photocatalytic activity, and for the fields of medical treatments, biological researches and the like.
  • a fusion protein comprising a peptide portion capable of binding to a titanium oxide and a peptide portion capable of binding to a carbon nanotube, it becomes possible to provide antimicrobial materials, deodorant materials, air cleaning materials, anti-stain materials, hydrogen generating apparatuses, solar cells, semiconductors and the like.
  • the present invention provides a fusion protein.
  • the fusion protein of the present invention can comprise a polypeptide portion capable of forming a multimer having an internal cavity, and a first peptide portion capable of binding to a first target substance and a second peptide portion capable of binding to a second target substance.
  • polypeptide portion capable of forming a multimer having an internal cavity refers to a polypeptide portion having an ability to form a multimer having a space inside thereof by association of the polypeptide portions.
  • proteins is known as such a polypeptide portion. Examples of such a polypeptide portion may include ferritin capable of forming a 24-meric structure having an internal cavity and a ferritin-like protein capable of forming a multimer having an internal cavity. Examples of the ferritin-like protein capable of forming the multimer having the internal cavity may include Dps capable of forming a 12-meric structure having an internal cavity.
  • the polypeptide portion capable of forming the multimer having the internal cavity may be naturally occurring proteins derived from any organism such as microorganisms, plants and animals or mutants of the naturally occurring proteins.
  • the polypeptide portion capable of forming the multimer having the internal cavity may be simply referred to as the polypeptide portion.
  • the polypeptide portion capable of forming the multimer having the internal cavity is Dps.
  • Dps DNA-binding protein from starved cells
  • the term “Dps” includes naturally occurring Dps or mutants thereof. For the mutants of the naturally occurring Dps, preferred are those exposing its N-terminal part and C-terminal part on the surface of the 12-meric structure upon formation of the 12-meric structure, as is similar to the naturally occurring Dps.
  • Dps may be also referred to as NapA, bacterioferritin, Dlp or MrgA depending on a type of the microorganism from which Dps is derived.
  • Subtypes such as DpsA, DpsB, Dps1 and Dps2 are also known for Dps (see, T. Haikarainen and A. C. Papageorgion, Cell. Mol. Life. Sci., 2010 vol. 67, p. 341). Therefore, the term “Dps” includes the proteins called by these other names.
  • the microorganism from which Dps is derived is not particularly limited as long as the microorganism produces Dps.
  • Examples of the microorganism may include bacteria belonging to genera Listeria, Staphylococcus, Bacillus, Streptococcus, Vibrio, Escherichia, Brucella, Borrelia, Mycobacterium, Campylobacter, Thermosynechococcus and Deinococcus , and Corynebacterium.
  • Examples of the bacteria belonging to genus Listeria may include Listeria innocua and Listeria monocytogenes .
  • Examples of the bacteria belonging to genus Staphylococcus may include Staphylococcus aureus .
  • Examples of the bacteria belonging to genus Bacillus may include Bacillus subtilis .
  • Examples of the bacteria belonging to genus Streptococcus may include Streptococcus pyogenes and Streptococcus suis .
  • Examples of the bacteria belonging to genus Vibrio may include Vibrio cholerae .
  • Examples of the bacteria belonging to genus Escherichia may include Escherichia coli .
  • Examples of the bacteria belonging to genus Brucella may include Brucella melitensis .
  • Examples of the bacteria belonging to genus Borrelia may include Borrelia burgdorferi .
  • Examples of the bacteria belonging to genus Mycobacterium may include Mycobacterium smegmetis .
  • Examples of the bacteria belonging to genus Campylobacter may include Campylobacter jejuni .
  • Examples of the bacteria belonging to genus Thermosynechococcus may include Thermosynechococcus elongates .
  • Examples of the bacteria belonging to genus Deinococcus may include Deinococcus radiodurans .
  • Examples of the bacteria belonging to genus Corynebacterium may include Corynebacterium glutamicum.
  • Dps may be a protein consisting of an amino acid sequence having 700 or more similarity to an amino acid sequence of Dps derived from Listeria innocua or Escherichia coli , or Corynebacterium glutamicum .
  • the percent similarity of the amino acid sequence of Dps to the amino acid sequence of Dps derived from Listeria innocua or Escherichia coli , or Corynebacterium glutamicum can be preferably 75% or more, more preferably 80% or more, still more preferably 85% or more, and most preferably 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • Dps has five ⁇ -helical segments in its secondary structure (see, A. Ilari et al., Nat. Struct. Biol., 2000, Vol. 7, p. 38, R. A. Grant et al. Nat. Struct. Biol. 1998, Vol. 5, p. 294, and R.
  • (ii) to (vi) can be important for retaining the ability to form the multimer having the internal cavity.
  • (i) and (ii) particularly (ii) can be important, since it is required that the ⁇ -helix adjacent to the N-terminal part of Dps faces outward the 12-meric structure.
  • (vi) and (vii) particularly (vi) can be important, since it is required that the ⁇ -helix adjacent to the C-terminal part of Dps faces outward the 12-meric structure.
  • any mutation may be introduced when an amino acid residue present in the regions other than the aforementioned important regions is mutated.
  • a person skilled in the art can easily prepare the mutant of the naturally occurring Dps by introducing a desired mutation into naturally occurring Dps so as to retain its function based on these guidelines.
  • the position of the amino acid residue to which the mutation is to be introduced in the amino acid sequence is apparent to a person skilled in the art as described above.
  • the mutant of naturally occurring Dps may be prepared further with reference to a sequence alignment.
  • a person skilled in the art can recognize correlation between structure and function because a person skilled in the art can 1) compare a plurality of amino acid sequences of Dps (e.g., the amino acid sequence represented by SEQ ID NO:4 and the amino acid sequence of other Dps), 2) demonstrate relatively conserved regions and relatively non-conserved regions, and then 3) predict regions capable of playing an important role for the function and regions incapable of playing an important role for the function from the relatively conserved regions and the relatively non-conserved regions, respectively.
  • a person skilled in the art can identify the position to which the mutation is to be introduced in the amino acid sequence of Dps by the aforementioned secondary structure alone, and also can identify the position of the amino acid residue to which the mutation is to be introduced in the amino acid sequence of Dps by combining the secondary structure information and the sequence alignment information.
  • the protein consisting of the amino acid sequence having 70% or more similarity to the amino acid sequence of Dps derived from Listeria innocua or Escherichia coli , or Corynebacterium glutamicum may be a protein consisting of an amino acid sequence that comprises one or several mutations of amino acid residues (e.g., deletions, substitutions, additions, and insertions) in the amino acid sequence of Dps derived from Listeria innocua or Escherichia coli , or Corynebacterium glutamicum , and retaining the function of Dps.
  • One or several mutations of the amino acid residues may be introduced into one region or a plurality of different regions in the amino acid sequence.
  • the number represented by the term “one or several” is, for example, 1 to 50, preferably 1 to 30, more preferably 1 to 20, still more preferably 1 to 10, and particularly preferably 1, 2, 3, 4 or 5.
  • substitution of the amino acid residue may be the conservative substitution.
  • conservative substitution refers to that a certain amino acid residue is substituted with an amino acid residue having a similar side chain. Families of the amino acid residues having the similar side chain are well-known in the art.
  • Examples of such a family may include amino acids having a basic side chain (e.g., lysine, arginine, histidine), amino acids having an acidic side chain (e.g., aspartic acid, glutamic acid), amino acids having a non-charged polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having a nonpolar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having a branched side chain at position ⁇ (e.g., threonine, valine, isoleucine), amino acids having an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan, histidine), amino acids having a hydroxyl group (alcoholic, phenolic)-containing side chain
  • the conservative substitution of the amino acids may be the substitution between aspartic acid and glutamic acid, the substitution among arginine, lysine and histidine, the substitution between tryptophan and phenylalanine, the substitution between phenylalanine and valine, the substitution among leucine, isoleucine and alanine, and the substitution between glycine and alanine.
  • the protein consisting of the amino acid sequence having 70% or more similarity to the amino acid sequence of Dps derived from Listeria innocua or Escherichia coli , or Corynebacterium glutamicum may be a protein encoded by a polynucleotide that hybridizes under a stringent condition with a nucleotide sequence complementary to a nucleotide sequence represented by SEQ ID NO:3 or SEQ ID NO:28, and retains the function of Dps.
  • the “stringent condition” refers to a condition where a so-called specific hybrid is formed whereas a non-specific hybrid is not formed.
  • such a condition is a condition where polynucleotides having high homology (e.g., identity or similarity), for example, 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more and particularly preferably 98% or more homology are hybridized with each other and polynucleotides having the lower homology than that are not hybridized.
  • polynucleotides having high homology e.g., identity or similarity
  • such a condition may include hybridization in 6 ⁇ SSC (sodium chloride/sodium citrate) at about 45° C. followed by washing once or twice or more with 0.2 ⁇ SSC and 0.1% SDS at 50 to 65° C.
  • Dps may be a protein consisting of an amino acid sequence having 70% or more identity to the amino acid sequence of Dps derived from Listeria innocua or Escherichia coli , or Corynebacterium glutamicum .
  • the percent identity of the amino acid sequences of Dps may be preferably 75% or more, more preferably 80% or more, still more preferably 85% or more, most preferably 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • Dps may be a protein consisting of, or comprising an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:29.
  • the percent identity of the amino acid sequence of the fusion protein of the present invention to the amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:29 may be preferably 95% or more, more preferably 96% or more, still more preferably 97% or more, particularly preferably 98% or more, or 99% or more.
  • the homology (e.g., identity or similarity) of the amino acid sequences and the nucleotide sequences can be determined, for example, using the algorithm BLAST (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) by Karlin and Altschul or FASTA (Methods Enzymol., 183, 63 (1990)) by Pearson.
  • the programs referred to as BLASTP and BLASTN were developed based on this algorithm BLAST (see http://www.ncbi.nlm.nih.gov).
  • the homology of the amino acid sequences and the nucleotide sequences may be calculated using these programs with default setting.
  • the lowest value among the values derived from these calculations may be employed as the homology of the nucleotide sequences and the amino acid sequences.
  • first peptide portion capable of binding to a first target substance and “second peptide portion capable of binding to a second target substance” refer to a portion that has a peptide having an affinity to any target substance and can bind to the target substance.
  • the first peptide portion and the second peptide portion may be the same or different from each other. Since various peptides having the affinity to the target substance are known, a portion having such a peptide can be used as the peptide portion in the present invention.
  • the first peptide portion and the second peptide portion may be simply referred to as the peptide portion capable of binding to the target substance.
  • the expression “the peptide portion capable of binding to the target substance” is an expression including the terms “the first peptide portion capable of binding to the first target substance” and “the second peptide portion capable of binding to the second target substance”, and thus, these expressions are interchangeably used.
  • the peptide portion capable of binding to the target substance may have only one peptide having the affinity to any target substance or may have a plurality of same or different peptides (e.g., several such as 2, 3, 4, 5, or 6) having the affinity to any target substance.
  • P1R5 peptide (SSKKSGSYSGSKGSKRRILGGGGHSSYWYAFNNKT [SEQ ID NO:21]) that is a fusion peptide of P1 peptide capable of binding to a carbon nanomaterial (SEQ ID NO:13) and R5 peptide capable of binding to a titanium material or a silicon material (SEQ ID NO:15) can be used as the peptide portion (see, e.g., M. J. Pender et al., Nano Lett., 2006, vol. 6, No. 1, p. 40-44).
  • the plurality of peptides can be fused in any order in the peptide portion.
  • the fusion can be accomplished via an amide bond.
  • the fusion can be accomplished directly via the amide bond or via the amide bond through a peptide (a peptide linker) consisting of one amino acid residue (e.g., methionine) or several (e.g., 2 to 50, preferably 2 to 30, more preferably 2 to 20, still more preferably 2 to 15 or 2 to 10, and most preferably 2, 3, 4, or 5) amino acid residues. Since various peptide linkers are known, such a peptide linker can also be used in the present invention.
  • target substance may include inorganic materials and organic materials, or conductive materials, semiconductor materials, and magnetic materials.
  • target substances may include metal materials, silicon materials, carbon materials, small compounds (e.g., biological substances such as porphyrin, radioactive substances, fluorescent substances, dyes, and medicines), polymers (e.g., hydrophobic organic polymers and conductive polymers such as poly(methyl methacrylate), polystyrene, polyethylene oxide, or poly(L-lactic acid)), proteins (e.g., oligopeptides or polypeptides), nucleic acids (DNA or RNA, or nucleosides, nucleotides, oligonucleotides or polynucleotides), carbohydrates (e.g., monosaccharides, oligosaccharides or polysaccharides), and lipids.
  • metal materials silicon materials, carbon materials, small compounds (e.g., biological substances such as porphyrin, radioactive substances, fluorescent substances, dyes, and medicines), polymers (e.
  • Examples of the metal materials may include metals and metal compounds.
  • Examples of the metal may include titanium, chromium, zinc, lead, manganese, calcium, copper, calcium, germanium, aluminium, gallium, cadmium, iron, cobalt, gold, silver, platinum, palladium, hafnium, and tellurium.
  • Examples of the metal compounds may include oxide, sulfide, carbonate, arsenide, chloride, fluoride and iodide of the metals, and intermetallic compounds.
  • Oxide of the metal may include various oxides. Describing such an oxide using the oxide of titanium as one example, examples of the oxide of titanium may include titanium monoxide (CAS No. 12137-20-1), titanium dioxide (CAS No.
  • the metal compounds may include oxides of titanium as described above, chromium oxide, zinc oxide, lead oxide, manganese oxide, zeolite, calcium carbonate, copper oxide, manganese-calcium oxide, germanium oxide, aluminium oxide, hafnium oxide, lead titanium zirconate, gallium arsenide, zinc sulfide, lead sulfide, cadmium sulfide, iron platinum, cobalt platinum, and cadmium tellurium.
  • Examples of the silicon materials may include silicon and silicon compounds.
  • Examples of the silicon compounds may include oxides of silicon (e.g., silicon monoxide (SiO), silicon dioxide (SiO 2 )), silicon carbide (SiC), silane (SiH 4 ), and silicone rubbers.
  • Examples of the carbon materials may include carbon nanomaterials (e.g., carbon nanotube (CNT), carbon nanohorn (CNH)), fullerene (C60), graphene sheet, and graphite.
  • carbon nanomaterials e.g., carbon nanotube (CNT), carbon nanohorn (CNH)
  • fullerene C60
  • graphene sheet graphite
  • the peptide portion capable of binding to the target substance is not particularly limited as long as it has an affinity to the target substance as described above.
  • Various peptides having the affinity to the target substance is known and developed.
  • the peptide capable of binding to the inorganic material or the organic material is developed by a technique such as screening using a phage for the purpose of making a complex of the biomaterial and the inorganic material or the organic material.
  • Examples of the peptides developed by such a technique may include peptides capable of binding to the metal materials such as titanium, oxides of titanium and silver (K. Sano et al., Langmuir, 2004, vol. 21, p. 3090, International Publication No. WO2005/010031), gold (S. Brown, Nat.
  • the peptide capable of binding to the metal is able to have an action for mineralization of the metal
  • the peptide capable of binding to the metal compound is able to have an action for mineralization of the metal compound
  • K. Sano et al., Langmuir, 2004, vol. 21, p. 3090, and M. Umetsu et al., Adv. Mater., 2005, vol. 17, p. 2571 K. Sano et al., Langmuir, 2004, vol. 21, p. 3090, and M. Umetsu et al., Adv. Mater., 2005, vol. 17, p. 2571. Therefore, when a peptide capable of binding to the metal material (metal or metal compound) is used as the peptide capable of binding to the target substance, the peptide capable of binding to the metal material can have such an action for the mineralization.
  • the fusion of the polypeptide portion and the first and second peptide portions can be accomplished via amide bonds.
  • the fusion can be accomplished directly via the amide bond or via the amide bond through a peptide (a peptide linker) consisting of one amino acid residue (e.g., methionine) or several (e.g., 2 to 50, preferably 2 to 30, more preferably 2 to 20, still more preferably 2 to 15 or 2 to 10, and most preferably 2, 3, 4, or 5) amino acid residues. Since various peptide linkers are known, such a peptide linker can also be used in the present invention.
  • a peptide linker consisting of one amino acid residue (e.g., methionine) or several (e.g., 2 to 50, preferably 2 to 30, more preferably 2 to 20, still more preferably 2 to 15 or 2 to 10, and most preferably 2, 3, 4, or 5) amino acid residues. Since various peptide linkers are known, such a peptide linker can also be used in the
  • the order of fusing the polypeptide portion and the first and second peptide portions in the fusion protein of the present invention is not particularly limited, 1) the N-terminal part and the C-terminal part of the polypeptide portion may be fused to the C-terminal part and the N-terminal part (or the N-terminal part and the C-terminal part) of the first and second peptide portions, respectively, or 2) the N-terminal part of the polypeptide portion may be fused to the C-terminal part of the first peptide portion and the N-terminal part of the first peptide portion may further be fused to the C-terminal part of the second peptide portion, or 3) the C-terminal part of the polypeptide portion may be fused to the N-terminal part of the first peptide portion and the C-terminal part of the first peptide portion may further be fused to the N-terminal part of the second peptide portion.
  • ferritin when ferritin is used as the polypeptide portion, ferritin is preferably fused in the order of 2) above, since the N-terminal part of ferritin is exposed on the surface of the multimer whereas the C-terminal part is not exposed on the surface.
  • Dps when Dps is used as the polypeptide portion, Dps may be fused in any of the orders of 1) to 3) above, since both the N-terminal part and the C-terminal part of Dps can be exposed on the surface of the multimer.
  • the fusion protein of the present invention can have the first peptide portion and the second peptide portion (one or plurality, respectively) on an N-terminal side and on a C-terminal side of the polypeptide portion, respectively.
  • the C-terminal part of the first peptide portion is fused to the N-terminal part of the polypeptide portion and the N-terminal part of the second peptide portion is fused to the C-terminal part of the polypeptide portion.
  • the first peptide portion can be designed so as to have methionine encoded by a translation initiation codon or a portion including methionine at its N-terminus on the N-terminal side of the first peptide portion.
  • the translation of the fusion protein of the present invention can be facilitated by such a design.
  • the peptide portion including methionine at the N-terminus may be a peptide consisting of several (e.g., 2 to 50, preferably 2 to 30, more preferably 2 to 20, still more preferably 2 to 15 or 2 to 10, and most preferably 2, 3, 4, or 5) amino acid residues.
  • the first peptide portion and the second peptide portion in the fusion protein of the present invention can bind to the different target substance.
  • a combination of the target substances to which the first peptide portion and the second peptide portion are bound may include a combination of the inorganic material and the organic material, a combination of two inorganic materials, and a combination of two organic materials. More specifically, such combinations may include a combination of the metal material and the silicon material, a combination of the metal material and the carbon material, a combination of the silicon material and the carbon material, a combination of two metal materials, a combination of two silicon materials, and a combination of two carbon materials. Therefore, the combination of the first peptide portion and the second peptide portion may be a combination of the peptide portions capable of binding to the target substances described above.
  • one of the first and second peptide portions may bind to the carbon material and the other may bind to the metal material or the silicon material in the fusion protein of the present invention.
  • the fusion protein of the present invention has a peptide portion capable of binding to the carbon material as the first peptide portion and has a peptide portion capable of binding to the metal material or the silicon material as the second peptide portion, or alternatively, has the peptide portion capable of binding to the metal material or the silicon material as the first peptide portion and has the peptide portion capable of binding to the carbon material as the second peptide portion.
  • a peptide portion capable of binding to the carbon material a peptide portion capable of binding to a carbon nanomaterial such as carbon nanotube (CNT) or carbon nanohorn (CNH) is preferred.
  • a carbon nanomaterial such as carbon nanotube (CNT) or carbon nanohorn (CNH)
  • Examples of such a peptide may include DYFSSPYYEQLF (SEQ ID NO:6) disclosed in Examples described later and JP Publication No. 2004-121154, HSSYWYAFNNKT (SEQ ID NO:13) disclosed in M. J. Pender et al., Nano Lett., 2006, vol. 6, No. 1, p. 40-44, and YDPFHII (SEQ ID NO:14) disclosed in JP Publication No. 2004-121154, or mutant peptides thereof (e.g., mutation such as conservative substitution for 1, 2, 3, 4 or 5 amino acid residues), or peptides having one or a plurality of such amino acid sequences.
  • a peptide portion capable of binding to the metal material a peptide portion capable of binding to a titanium material such as titanium or a titanium compound (e.g., a titanium oxide), and a peptide portion capable of binding to a zinc material such as zinc or a zinc compound (e.g., a zinc oxide) are preferred.
  • a titanium material such as titanium or a titanium compound (e.g., a titanium oxide)
  • a zinc material such as zinc or a zinc compound (e.g., a zinc oxide)
  • examples of the peptide portion capable of binding to the titanium material may include RKLPDA (SEQ ID NO:8) disclosed in Examples described later and International Publication No. WO2006/126595, SSKKSGSYSGSKGSKRRIL (SEQ ID NO:15) disclosed in M. J. Pender et al., Nano Lett., 2006, vol. 6, No. 1, p.
  • RKLPDAPGMHTW SEQ ID NO:16
  • RALPDA SEQ ID NO:17
  • mutant peptides thereof e.g., mutation by conservative substitution of 1, 2, 3, 4 or 5 amino acid residues
  • peptides having one or several such an amino acid sequence examples include EAHVMHKVAPRPGGGSC (SEQ ID NO:30) disclosed in Example described later and Umetsu et al., Adv. Mater., 17, 2571-2575 (2005), or mutant peptides thereof (e.g., mutation such as conservative substitution for 1, 2, 3, 4 or 5 amino acid residues), or peptides having one or a plurality of such amino acid sequences.
  • a peptide portion capable of binding to silicon or a silicon compound e.g., an oxide of silicon
  • examples of such a peptide portion may include RKLPDA (SEQ ID NO:8) disclosed in Examples described later and International Publication No. WO2006/126595, SSKKSGSYSGSKGSKRRIL (SEQ ID NO:15) disclosed in M. J. Pender et al., Nano Lett., 2006, vol. 6, No. 1, p. 40-44, and MSPHPHPRHHHT (SEQ ID NO:18), TGRRRRLSCRLL (SEQ ID NO:19) and KPSHHHHHTGAN (SEQ ID NO:20) disclosed in International Publication No. WO2006/126595, or mutant peptides thereof (e.g., mutation such as conservative substitution for 1, 2, 3, 4 or 5 amino acid residues), or peptides having one or a plurality of such amino acid sequences.
  • the fusion protein of the present invention may be a protein consisting of, or comprising an amino acid sequence having 90% or more identity to an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:27, or SEQ ID NO:32.
  • the percent identity of the amino acid sequence of the fusion protein of the present invention to the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:27, or SEQ ID NO:32 may be preferably 95% or more, more preferably 96% or more, still more preferably 97% or more, and particularly preferably 98% or more or 99% or more.
  • the fusion protein of the present invention can be obtained from a transformant that expresses the fusion protein of the present invention.
  • This transformant can be prepared by making an expression vector for the fusion protein of the present invention comprising a polynucleotide encoding the fusion protein of the present invention and then introducing this expression vector into a host.
  • Examples of the host for expressing the fusion protein of the present invention may include various prokaryotic cells including bacteria belonging to genera Escherichia ( Escherichia coli ) and Corynebacterium , and Bacillus subtilis , and various eukaryotic cells including Saccharomyces cerevisiae, Pichia stipitis and Aspergillus oryzae.
  • E. coli as the host to be transformed will be described in detail.
  • Examples of E. coli may include E. coli JM109 strain, DH5 ⁇ strain, HB101 strain, and BL21 (DE3) strain that are subtypes of E. coli K12 strain. Methods of transformation and methods of selecting the transformant are described in Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press (2001 Jan. 15). A method of making transformed E. coli and producing the fusion protein of the present invention using this will be specifically described below as one example.
  • a promoter for expressing a DNA encoding the fusion protein of the present invention a promoter generally used for the production of a heterologous protein in E. coli can be used.
  • the promoter may include strong promoters such as a T7 promoter, a lac promoter, a trp promoter, a trc promoter, a tac promoter, a PR promoter and a PL promoter of lambda phage, and a T5 promoter.
  • Examples of a vector may include pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pACYC177, pACYC184, pMW119, pMW118, pMW219, pMW218, pQE30, and derivatives thereof.
  • a terminator that is a transcription termination sequence may be ligated downstream of a gene encoding the fusion protein of the present invention.
  • Examples of such a terminator may include a T7 terminator, a fd phage terminator, a T4 terminator, a terminator of a tetracycline resistant gene, and a terminator of an E. coli trpA gene.
  • the vector for introducing a gene encoding the fusion protein of the present invention is preferably a so-called multicopy type, and may include a plasmid having a replication origin derived from ColE1, e.g., pUC-based plasmids and pBR322-based plasmids or derivatives thereof.
  • the “derivative” means a plasmid modified by substitution, deletion, insertion, addition, and/or inversion of a nucleotide(s).
  • the “modification” referred to herein includes a mutation treatment by a mutating agent or irradiation with UV or the modification by natural mutation.
  • the vector preferably has a marker such as an ampicillin resistant gene for selecting the transformant.
  • the expression vectors having the strong promoter are commercially available (e.g., pUC-based vectors manufactured by Takara Bio Inc., pPROK-based vectors manufactured by Clontech, and pKK233-2 manufactured by Clontech).
  • the fusion protein of the present invention is expressed.
  • culture media may include media such as M9-casamino acid medium and LB medium generally used for culturing E. coli .
  • Conditions on cultivation, production induction and the like can be selected appropriately depending on the types of the marker in the vector used, the promoter, the host microorganism and the like.
  • the fusion protein of the present invention can be obtained as a disrupted product or a lysed product by collecting the transformant that produces the fusion protein of the present invention followed by disrupting (e.g., sonication or homogenization) or lysing (e.g., a lysozyme treatment) the transformant.
  • a purified protein, a crude purified protein, or a fraction containing the fusion protein of the present invention can be obtained by subjecting such a disrupted product or lysed product to a technique such as extraction, precipitation, filtration, or column chromatography.
  • the present invention also provides a polynucleotide encoding the fusion protein of the present invention and an expression vector comprising the polynucleotide and a transformant comprising the expression vector, as described above, which can be used for preparing the fusion protein of the present invention.
  • the polynucleotide of the present invention can comprise a polynucleotide portion encoding the polypeptide portion capable of forming the multimer having the internal cavity, and a polynucleotide portion encoding the first peptide portion capable of binding to the first target substance, and a polynucleotide portion encoding the second peptide portion capable of binding to the second target substance.
  • the polynucleotide of the present invention can be specified from various points based on the aforementioned descriptions on the fusion protein of the present invention, since it encodes the fusion protein of the present invention.
  • the polynucleotide of the present invention may be a polynucleotide consisting of, or comprising a nucleotide sequence having 90% or more identity to a nucleotide sequence represented by SEQ ID NO:1, SEQ ID NO:26, or SEQ ID NO:31.
  • the percent identity of the nucleotide sequence of the polynucleotide of the present invention to the nucleotide sequence represented by SEQ ID NO:1, SEQ ID NO:26, or SEQ ID NO:31 may be preferably 95% or more, more preferably 96% or more, still more preferably 97% or more, and particularly preferably 98% or more or 99% or more.
  • the present invention provides a multimer of the fusion protein.
  • the multimer of the present invention can have the internal cavity.
  • the fusion protein that composes the multimer of the present invention is as described above.
  • the multimer of the present invention can be formed autonomously by expressing the fusion protein of the present invention.
  • the number of monomer units that compose the multimer of the present invention can be determined by the type of the polypeptide portion in the fusion protein of the present invention.
  • the multimer of the present invention may be a 12-meric structure, since it may have Dps as the polypeptide portion capable of forming the multimer having the internal cavity.
  • the multimer of the present invention may be a homomultimer composed of a single fusion protein as the monomer unit or may be a heteromultimer composed of a plurality of different fusion proteins (e.g., 2, 3, 4, 5, or 6).
  • the polypeptide portion in the fusion protein that composes the multimer is preferably a single polypeptide portion in terms of forming the multimer, but the first peptide portion and the second peptide portion may be different in the fusion proteins that compose the multimer.
  • examples of a combination of two types of the fusion proteins may include the followings:
  • the multimer of the present invention is composed of two types of the fusion proteins and at least a peptide portion capable of binding to the carbon material and a peptide portion capable of binding to the metal material (e.g., titanium material, zinc material) or the silicon material are used as the peptide portions
  • the metal material e.g., titanium material, zinc material
  • examples of the combination of two types of the fusion proteins may include the followings:
  • the multimer composed of a plurality of different types of the fusion proteins can be obtained by, for example, introducing a plurality of vectors expressing the different types of the fusion proteins or a single vector expressing the different types of the fusion proteins (e.g., vector capable of expressing polycistronic mRNA) into a single host cell and then expressing the different types of the fusion proteins in the single host cell.
  • a multimer can also be obtained by allowing a first monomer composed of a single fusion protein and a second monomer composed of a single fusion protein (different from the fusion protein that composes the first monomer) to coexist and be left stand in the same vehicle (e.g., buffer).
  • the monomer of the fusion protein can be prepared by, for example, leaving stand the multimer of the present invention in buffer at low pH.
  • the monomer of the fusion protein can be prepared by, for example, leaving stand the multimer of the present invention in buffer at low pH.
  • the multimer of the present invention may contain a substance in its internal cavity.
  • the substance in a form of a complex or a particle e.g., nanoparticle, magnetic particle
  • a person skilled in the art can appropriately select a substance that can be encapsulated in the multimer of the present invention by considering a size of the internal cavity of the multimer of the present invention, a charge property of the amino acid residues in regions involved in encapsulation of the substance in the multimer of the present invention (e.g., C-terminal region: see R. M. Kramer et al., 2004, J. Am. Chem. Soc., vol. 126, p. 13282), and the like.
  • the size of the substance that can be encapsulated in such a multimer can be, for example, 60 nm 3 or less, preferably 40 nm 3 or less, more preferably 20 nm 3 or less, still more preferably 10 nm 3 or less, and most preferably 5 nm 3 or less.
  • the encapsulation of the substance into the internal cavity of the multimer can further be facilitated by changing the charge property in the region that can be involved in the encapsulation of the substance in the multimer (e.g., type and number of amino acid residues having a side chain that can be charged positively or negatively) (see, e.g., R. M. Kramer et al., 2004, J. Am. Chem. Soc., vol. 126, p. 13282). Therefore, the multimer of the fusion protein having the region in which the charge property is changed can also be used in the present invention.
  • Examples of the substance that can be encapsulated in the multimer of the present invention may include inorganic materials as is similar to the aforementioned target substances.
  • the substances that can be encapsulated in the multimer of the present invention may include the metal materials and the silicon materials as described above. More specifically, such a substance may include iron oxides, nickel, cobalt, manganese, phosphorus, uranium, beryllium, aluminium, cadmium sulfide, cadmium selenide, palladium, chromium, copper, silver, gadolium complex, platinum cobalt, silicon oxide, cobalt oxide, indium oxide, platinum, gold, gold sulfide, zinc selenide, and cadmium selenium.
  • the encapsulation of the substance in the internal cavity of the multimer of the present invention can be carried out by known methods. For example, it can be carried out in the same manner as in the method of encapsulating the substance in the internal cavity of the multimer of ferritin or a ferritin-like protein such as Dps (see, e.g., I. Yamashita et al., Chem., Lett., 2005. vol. 33, p. 1158).
  • the substance can be encapsulated in the internal cavity of the multimer of the present invention by allowing the multimer of the present invention (or fusion protein of the present invention) and the substance to be encapsulated to coexist in the buffer such as HEPES buffer and then leaving them stand at an appropriate temperature (e.g., 0 to 37° C.) (see also Example 3).
  • the buffer such as HEPES buffer
  • the multimer of the present invention may be provided as a set of a plurality of different types of the multimers containing a plurality (e.g., 2, 3, 4, 5 or 6) of different types of the substances when containing the substance in the internal cavity.
  • a plurality e.g. 2, 3, 4, 5 or 6
  • the multimer of the present invention is provided as a set of two types of the multimers containing two types of the substances, such a set can be obtained by combining a first multimer encapsulating a first substance and a second multimer encapsulating a second substance (different from the first substance), which are each prepared separately.
  • Highly diverse multimers of the present invention can be obtained by appropriately combining diversified patterns of the fusion proteins with diversified patterns of the substances to be encapsulated, as described above.
  • the present invention also provides a complex.
  • the complex of the present invention can comprise the multimer of the present invention and the first and/or second target substance.
  • the first target substance can bind to the first peptide portion in the fusion protein
  • the second target substance can bind to the second peptide portion in the fusion protein.
  • the target substances are as described above.
  • the first and the second target substances are preferably different target substances.
  • the target substance may bind to another substance or object.
  • the target substance may be fixed onto a solid phase (e.g., plates such as well plates, supports, substrates, elements, and devices). Therefore, the complex of the present invention may further comprise the other substance or object as long as it can comprise the multimer of the present invention and the first target substance and/or the second target substance.
  • a porous structure can be prepared by burning the complex of the present invention.
  • the porous structure is described a porous structure in which a first empty hole has been formed in a site where the multimer of the present invention was present can be obtained by, for example, burning the complex of the present invention at temperature at which the protein can be extinguished.
  • a second empty hole indicates an empty hole that can be produced in a process of the precipitation of the second target substance and/or the burning.
  • a porous structure in which the first empty hole has been formed in the site where the multimer of the present invention was present and a third empty hole has been formed in a site where the first target substance was present can be obtained by burning the complex of the present invention at temperature at which both the protein and the first target substance (e.g., carbon material such as carbon nanotube) that composes an aggregate can be extinguished.
  • the substance e.g., metal particle
  • the substance e.g., metal particle
  • the porous structure thus produced is useful for the development of the devices, the materials and the like that are excellent in photocatalytic activity and electric properties.
  • the porous structure is useful as a material or a constituent for producing photoelectric conversion elements (e.g., solar cells such as dye sensitized solar cells), hydrogen generation elements, water cleaning materials, antimicrobial materials, and semiconductor memory elements.
  • the metal-encapsulating protein Dps from Listeria innocua an N-terminus of which is fused with a carbon nanohorn-binding protein (abbreviated as CNHBP and consisting of the amino acid sequence DYFSSPYYEQLF (SEQ ID NO:6); see International Publication No. WO2006/068250) and a C-terminus of which is fused with a titanium oxide-binding protein (abbreviated as TBP and consisting of the amino acid sequence RKLPDA (SEQ ID NO:8); see International Publication No. WO2005/010031) was constructed (abbreviated as CNHBP-Dps-TBP or CDT, SEQ ID NOS:1 and 2) by the following procedure.
  • CNHBP carbon nanohorn-binding protein
  • TBP titanium oxide-binding protein
  • RKLPDA amino acid sequence RKLPDA
  • synthesized DNAs (SEQ ID NO:9 and SEQ ID NO:10) was annealed by heating a mixed solution of the synthesized DNAs at 98° C. for 30 seconds and rapidly cooling it to 4° C.
  • This DNA solution and pET20 carrying a Dps gene from Listeria innocua were separately digested completely with a restriction enzyme NdeI.
  • the PCR product digested with the restriction enzymes was self-ligated using the T4 DNA ligase (Promega, USA).
  • E. coli JM109 (Takara Bio Inc., Japan) was transformed with the self-ligated PCR product to construct JM 109 possessing the expression plasmid (pET20-CDT) carrying the gene encoding Dps (CDT), the N-terminus of which was fused with the carbon nanohorn-binding peptide and the C-terminus of which was fused with the titanium-binding peptide.
  • the plasmid pET20-CDT was purified from the transformant using Wizard Plus Miniprep System (Promega, USA).
  • BL21 (DE3) (Invitrogen, USA) was transformed with pET20-CDT to make a strain BL21 (DE3)/pET20-CDT for expressing the protein. Meanwhile, a strain for expressing CD used for control experiments was produced similarly.
  • BL21 (DE3)/pET20-CDT were cultured in 5 mL of LB medium (containing 100 mg/L of ampicillin) at 37° C. Eighteen hours after starting the cultivation, the cultured medium was inoculated to 3 L of new LB medium (containing 100 mg/L of ampicillin) and cultured with shaking using BMS-10/05 (ABLE, Japan) at 37° C. for 24 hours. The resulting microbial cells were collected by centrifugation (5,000 rpm, 5 minutes), and stored at ⁇ 80° C. A half (6 g) of the cryopreserved microbial cells was suspended in 40 mL of 50 mM Tris-HCl buffer (pH 8.0).
  • the microbial cells were disrupted by giving an ultrasonic pulse (200 W, Duty 45%) to the suspension every one second for 12 minutes using Digital Sonifier 450 (Branson, USA).
  • the solution was centrifuged at 15,000 rpm for 15 minutes (JA-20, Beckman Coulter, USA), and a supernatant fraction was collected.
  • the collected solution was heated at 60° C. for 20 minutes, and rapidly cooled on ice after the heating.
  • the cooled solution was centrifuged (JA-20) at 17,000 rpm for 10 minutes, and a supernatant (about 20 mL) was collected again.
  • This solution was sterilized using a disc filter (Millex GP, 0.22 ⁇ m, Millipore, USA). And, this solution was ultrafiltrated and concentrated using Amicon-Ultra-15 (NMWL. 50000, Millipore, USA) until a liquid amount became 10 mL to obtain a protein solution.
  • a CDT fraction that was an objective protein was purified from the protein solution using gel filtration chromatography. Specifically, 10 mL of the protein solution was applied to HiPrep 26/60 Sephacryl S-300 High resolution column (GE Healthcare, USA) equilibrated with Tris-HCl buffer (50 mM Tris-HCl solution containing 150 mM NaCl, pH 8.0), and separated/purified at a flow rate of 1.4 mL/minute to collect fractions corresponding to CDT. The following experiments were carried out using the purified CDT. Concerning CD used for the control experiments, a gene was expressed in the same manner as in CDT, the microbial cells were collected and treated with heat.
  • an iron oxide nanoparticle was formed in an internal cavity of the CDT multimer. Specifically, 1 mL of HEPES buffer (80 mM HEPES/NaOH (pH 7.5), 0.5 mg/mL of CDT, 1 mM iron ammonium sulfate, each at final concentration) containing CDT was prepared and left stand at 4° C. for 3 hours.
  • CDT after forming the iron oxide nanoparticle was stained with PTA and observed, and the iron oxide nanoparticle having a diameter of about 5 nm was formed in the internal cavity of the CDT multimer with an outer diameter of about 9 nm. Also, in the staining with Au-Glc, it could be observed that the iron oxide nanoparticle was formed in this internal cavity.
  • CDT before forming the iron oxide nanoparticle was stained with PTA and observed under the electron microscope, and a globular protein alone having an outer diameter of about 9 nm was observed.
  • CNHBP carbon nanohorn-binding peptide fused to the N-terminus of CDT. It is known that CNHBP recognizes not only the carbon nanohorn (CNH) but also the carbon nanotube (CNT) (see International Publication No. WO2006/068250).
  • HEPES buffer containing CDT or Dps (20 mM HEPES/NaOH (pH7.5), 0.3 mg/mL of CDT or Dps, and 0.3 mg/mL CNT (Sigma-Aldrich, 519308, carbon nanotube, single walled) each at final concentration) was prepared.
  • the ultrasonic pulse every one second was given to this solution for 5 minutes using Digital Sonifier 450 (Branson, USA).
  • the ultrasonicated protein/CNT mixed solution was centrifuged (15,000 rpm, 5 minutes), and complexes of the protein and CNT contained in the supernatant were stained with 3% PTA and observed under the transmission electron microscope (JEM-2200FS, 200 kV).
  • TBP titanium oxide-binding peptide
  • the surface of the titanium sensor was washed by placing 50 ⁇ L of a washing solution [in which 98% (w/v) sulfuric acid and 30% (w/v) hydrogen peroxide water were mixed at 3:1] on the titanium sensor for the measurement for one minute and then washing it out with water. After repeating this washing three times, the titanium sensor was attached to a main body (QCM934, SEIKO EG and G). A frequency value of the titanium sensor was stabilized by dropping 500 ⁇ L of TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) on the sensor and leaving it stand at room temperature for 3 hours.
  • TBS buffer 50 mM Tris-HCl, 150 mM NaCl, pH 8.0
  • TEMOS tetramethyloxysilane
  • Shin-ETsu Silicone tetramethyloxysilane
  • the aqueous solution of TEMOS was prepared by mixing 143 ⁇ L of 1 mM HCl and 25 ⁇ L of TEMOS liquid well, leaving it stand at room temperature for 5 minutes, and then diluting it with the TBS buffer to 10 times.
  • the aqueous solution of TEMOS was applied onto the sensor, the frequency was reduced and mineralization of silicon oxide was observed.
  • the sensor on which silicon oxide had been mineralized was washed with the TBS buffer, and then the protein solution was applied thereon.
  • the frequency was reduced when the CDT solution was applied whereas the frequency was not reduced when the CD solution was applied. That is, it was suggested that CD having no TBP could not bind to silicon oxide whereas CDT having TBP could bind to silicon oxide.
  • TBP presented on CDT retained an ability to bind to titanium oxide and silicon oxide.
  • Amino acid sequences of Dps derived from other microorganisms were subjected to the analysis of homology to the amino acid sequences of Dps derived from Listeria innocua and Escherichia coli .
  • the homology was analyzed using the genetic information analysis software Genetyx (Genetyx Corporation). Algorithm of this software was based on Lipman-Pearson method (Lipman, D. J. and Pearson, W. R. 1985. Rapid and sensitive protein similarity searches. Science 227:1435-1441.) Results of analyzing the homology (identity and similarity) to the amino acid sequence of Dps derived from Listeria innocua are shown in Table 2.
  • PCR was carried out using pET20-CDT as a template DNA, and oligonucleotides consisting of the following nucleotide sequences as the primers.
  • the resulting PCR product was purified by Wizard SV Gel and PCR Clean-Up System (Promega, USA), and digested with restriction enzymes DpnI and NdeI.
  • the PCR product digested with the restriction enzymes was self-ligated using the T4 DNA ligase (Promega, USA).
  • E. coli JM109 (Takara Bio Inc., Japan) was transformed with the self-ligated PCR product to construct JM109 possessing an expression plasmid carrying the gene encoding DT (pET20-DT).
  • the plasmid pET20-DT was purified from the transformant using Wizard Plus Minipreps System (Promega, USA).
  • BL21 (DE3) Invitrogen, USA
  • pET20-DT was transformed with pET20-DT to obtain a strain BL21 (DE3)/pET20-DT for expressing protein.
  • DT was expressed in the same manner as in the case of CDT.
  • BL21 (DE3)/pET20-CDT, and BL21 (DE3)/pET20-DT and BL21 (DE3)/pET20-CD were cultured in 1 mL of LB medium (containing 100 mg/mL of ampicillin) at 37° C., respectively. Eighteen hours after starting the cultivation, the resulting culture medium was inoculated to 100 mL of new LB medium (containing 100 mg/L of ampicillin) and cultured with shaking using a 500 mL flask at 37° C. for 24 hours.
  • the resulting microbial cells were collected by centrifugation (6,000 rpm, 5 minutes), and suspended in 5 mL of 50 mM Tris-HCl buffer (pH 8.0). The microbial solution was sonicated to disrupt the microbial cells. The resulting solution was centrifuged at 6,000 rpm for 15 minutes and a supernatant fraction was collected. The collected solution was heated at 60° C. for 20 minutes and then rapidly cooled on ice. The cooled solution was centrifuged at 6,000 rpm for 15 minutes and a supernatant (about 5 mL) was collected again. This solution was sterilized using the disc filter (Millex GP. 0.22 ⁇ m, Millipore, USA).
  • the solution was ultrafiltrated and concentrated using Amicon-Ultra-15 (NMWL. 50000, Millipore, USA), and the buffer in which the protein was dissolved was replaced with Tris-HCl salt buffer (50 mM Tris-HCl solution containing 150 mM NaCl, pH 8.0) to obtain 2.5 mL of a protein solution.
  • Tris-HCl salt buffer 50 mM Tris-HCl solution containing 150 mM NaCl, pH 8.0
  • CDT, CD and DT were purified from the obtained protein solutions.
  • gel filtration and anion exchange chromatography were used for the purification of CDT and DT.
  • 2.5 mL of the crude extraction solution was applied to HiPrep 26/60 Sephacryl S-300 High resolution column (GE healthcare, USA) equilibrated with Tris-HCl salt buffer (50 mM Tris-HCl solution containing 150 mM NaCl, pH 8.0), eluted at a flow rate of 1.4 mL/minute, and fractions corresponding to each protein were collected.
  • Tris-HCl salt buffer 50 mM Tris-HCl solution containing 150 mM NaCl, pH 8.0
  • fractions corresponding to each protein were collected.
  • each the protein solution obtained was ultrafiltrated and concentrated, and the buffer of the protein solution was replaced with 50 mM Tris-HCl buffer (pH 8.0).
  • a binding rate constant and a dissociation rate constant between CDT and CNT were measured by QCM method.
  • the surface of a gold sensor was washed by placing 50 ⁇ L of a washing solution [in which 98% (w/v) sulfuric acid and 30% (w/v) hydrogen peroxide water were mixed at 3:1] on the gold sensor for the measurement for 5 minutes and then washing it out with water.
  • CNT carbon nanotube, single-walled, 519308, Aldrich
  • confirmed to form a complex with the protein under the transmission electron microscope was mixed at a final concentration of 1 mg/L with a 1% SDS solution and the mixture was sonicated for 30 minutes.
  • phosphate buffer A 50 mM potassium phosphate buffer containing 0.001% (w/v) Tween-20, pH 7.4
  • This CNT sensor was attached on the main body (Affinix, QN ⁇ , Initium), and the frequency value of the sensor was stabilized by dropping the phosphate buffer A on the CNT sensor, which was then left stand for 30 minutes to one hour.
  • the binding rate constant and the dissociation rate constant between CDT and titanium oxide were measured by QCM method.
  • a titanium oxide sensor manufactured by Inisium was used. First, a 1% SDS solution was placed on the titanium oxide sensor, the sensor was washed by pipetting, and the extra SDS solution was washed out five times with water. This washing was repeated twice. Further, the sensor was washed once with phosphate buffer B (50 mM phosphate buffer pH 7.0). This titanium oxide sensor was attached on the main body (Affinix, QN ⁇ , Initium), and the frequency value of the sensor was stabilized by dropping the phosphate buffer B on the sensor and leaving it stand at room temperature for 30 minutes to one hour.
  • phosphate buffer B 50 mM phosphate buffer pH 7.0
  • a mutant protein having the similar nature as that of CDT was constructed using Dps derived from Corynebacterium glutamicum .
  • the results of analysis of the amino acid sequence of Dps derived from Corynebacterium glutamicum to the amino acid sequences of Dps derived from Listeria innocua and Escherichia coli (identity and similarity) are shown in Table 7.
  • PCR was carried out using genomic DNA from Corynebacterium glutamicum as the template, and oligonucleotides consisting of the following nucleotide sequences as the primers.
  • the obtained PCR product was purified using Wizard SV Gel and PCR Clean-Up System (Promega, USA) and digested with the restriction enzymes NdeI and EcoRI. Meanwhile, the plasmid pET20b (Merck, Germany) was digested with the restriction enzymes NdeI and BamHI. The PCR product and the plasmid digested with the restriction enzymes were ligated using the T4 ligase (Promega, USA). E.
  • JM109 (Takara Bio Inc., Japan) was transformed with the resulting DNA to construct JM109 possessing an expression plasmid (pET20-CcDT) carrying the gene encoding Dps derived from Corynebacterium glutamicum , the N-terminus of which was fused with the carbon nanohorn-binding peptide (CNHBP), and the C-terminus of which was fused with the titanium-binding peptide (TBP) (CcDT, SEQ ID NOS:26 and 27).
  • CNHBP carbon nanohorn-binding peptide
  • TBP titanium-binding peptide
  • CcDT titanium-binding peptide
  • the plasmid pET20-CcDT was purified from this transformant using Wizard Plus Minipreps System (Promega, USA).
  • BL21 (DE3) (Invitrogen, USA) was transformed with pET20-CcDT to obtain the strain BL21 (DE3)/pET20-CcDT for expressing the
  • BL21 (DE3)/pET20-CcDT was cultured in 1 mL of LB medium (containing 100 mg/L of ampicillin) at 37° C. Eighteen hours after starting the cultivation, the resulting culture medium was inoculated to 100 mL of new LB medium (containing 100 mg/L of ampicillin), and cultured with shaking using a 500 mL flask at 37° C. for 24 hours. The resulting microbial cells were collected by centrifugation (6,000 rpm, 5 minutes), and suspended in 5 mL of 50 mM Tris-HCl buffer (pH 8.0). The microbial solution was sonicated to disrupt the microbial cells.
  • the resulting solution was centrifuged at 6,000 rpm for 15 minutes and a supernatant fraction was collected.
  • the collected solution was heated at 60° C. for 20 minutes and then rapidly cooled on ice.
  • the cooled solution was centrifuged at 6,000 rpm for 15 minutes and a supernatant (about 5 mL) was collected again.
  • This solution was sterilized using the disc filter (Millex GP. 0.22 Millipore, USA). And, the solution was ultrafiltrated and concentrated using Amicon-Ultra-15 (NMWL. 50000, Millipore, USA), and the buffer in which the protein was dissolved was replaced with Tris-HCl buffer (50 mM Tris-HCl pH 8.0) to obtain 2.5 mL of a protein solution.
  • anion exchange chromatography was used to purify CcDT from the resulting protein solution. Specifically, 2.5 mL of the protein solution was applied to HiLoard 26/10 Q-Sepharose High Performance column (GE healthcare, USA) equilibrated with 50 mM Tris-HCl buffer (pH 8.0). The separation/purification was carried out at a flow rate of 4.0 mL/minute by making a concentration gradient of the salt from 0 mM to 500 mM NaCl in 50 mM Tris-HCl buffer (pH 8.0), and fractions containing CcDT were collected.
  • HiLoard 26/10 Q-Sepharose High Performance column GE healthcare, USA
  • the separation/purification was carried out at a flow rate of 4.0 mL/minute by making a concentration gradient of the salt from 0 mM to 500 mM NaCl in 50 mM Tris-HCl buffer (pH 8.0), and fractions containing CcDT were collected.
  • CcDT was stained with 3% PTA (phosphotungstic acid), and analyzed under the transmission electron microscope. As a result, it was found that CcDT formed a cage-like multimer having a diameter of about 9 nm, as is similar to CDT.
  • the binding between CcDT and CNT was measured by QCM method.
  • the surface of the gold sensor was washed by placing 50 ⁇ L of a washing solution [in which 98% (w/v) sulfuric acid and 30% (w/v) hydrogen peroxide water were mixed at 3:1] on the gold sensor for the measurement for five minutes and then washing it out with water.
  • CNT was mixed at a final concentration of 1 mg/mL with 1% SDS solution, and the mixture was sonicated for 30 minutes to prepare a CNT solution. 2 ⁇ L of this CNT solution was mounted on the gold electrode and dried naturally at room temperature. After the drying, the sensor was washed twice with water to wash out CNT not bound to the sensor.
  • the sensor was further washed once with phosphate buffer A (50 mM potassium phosphate buffer containing 0.001% (w/v) Tween 20, pH 7.0).
  • phosphate buffer A 50 mM potassium phosphate buffer containing 0.001% (w/v) Tween 20, pH 7.0.
  • This CNT sensor was attached to the main body (Affinix QN ⁇ , Initium), and the frequency value was stabilized by dropping the phosphate buffer A on the CNT sensor and leaving it stand at room temperature for 30 minutes to one hour. After stabilizing the frequency value, 500 ⁇ L of a reaction solution containing CcDT at a final concentration of 1 mg/L was added, and the change of the frequency was measured.
  • CcDT having CNHBP could bind to CNT more abundantly than DT having no CNHBP. Therefore, it was found that CcDT has the stronger ability to bind to CNT than DT. It was speculated that this ability of CcDT to bind to CNT depended on CNHBP.
  • the binding between CcDT and titanium oxide was measured by QCM method.
  • the surface of the titanium oxide sensor was washed by placing 50 ⁇ L of the washing solution [in which 98% (w/v) sulfuric acid and 30% (w/v) hydrogen peroxide water were mixed at 3:1] on the sensor for the measurement for five minutes and then washing it out with water.
  • the titanium oxide sensor was attached to the main body (Affinix QN ⁇ , Initium), and the frequency value was stabilized by dropping the phosphate buffer B (50 mM potassium phosphate buffer, pH 7.0) on the titanium oxide sensor and leaving it stand at room temperature for 30 minutes to one hour. After stabilizing the frequency value, 500 ⁇ L of a reaction solution containing CcDT was added at a final concentration of 1 mg/L, and the change of the frequency was measured.
  • the phosphate buffer B 50 mM potassium phosphate buffer, pH 7.0
  • an iron oxide nanoparticle was formed in an internal cavity of a CDT multimer. Specifically, 1 mL of HEPES buffer containing CDT (80 mM HEPES/NaOH, pH 7.5 containing 0.5 mg/mL of CDT and 1 mM ammonium iron sulfate each at a final concentration) was prepared, and left stand at 4° C. for 3 hours. Then, this buffer was centrifuged (15,000 rpm, 5 minutes), and a supernatant containing the protein was collected. This supernatant was ultrafiltrated and concentrated using Amicon-Ultra-15 (NMWL.
  • NMWL Amicon-Ultra-15
  • the Fe-CDT-CNT mixed solution treated by the ultrasonic pulse was centrifuged (15,000 rpm, 5 minutes) to obtain a CNT/Fe-CDT complex in which many CDT multimers were bound to CNT.
  • the result is shown from a transmission electron microscopic image of the complex of CNT and CNHBP-Dps-TBP (CDT multimer) having the iron oxide nanoparticle in its internal cavity.
  • the transmission electron microscopic image was obtained by photographing the sample stained with 3% TPA.
  • the titanium precursor, titanium (IV) bis(ammonium lactato)dihydroxide (Sigma, 388165) was added to the obtained CNT/Fe-CDT complex solution such that the final concentration of the precursor became 2.5% by weight, and the mixture was left stand at room temperature (24° C.)
  • a transmission electron microscopic image of a black precipitate obtained by adding the titanium precursor to the complex of CNT and CNHBP-Dps-TBP shows the iron oxide nanoparticle in its internal cavity.
  • This black rod-shaped structure could be speculated to contain titanium from the result of EDS analysis.
  • 64 CDT multimers were encapsulated in the titanium nano-rod structure having a length of 102 nm in a lengthwise direction, and a diameter of 31 nm in a direction orthogonal to the lengthwise direction.
  • the surface area of the titanium nano-rod structure is 1.1 ⁇ 10 4 nm 2 .
  • the surface area of the CDT multimer having a diameter of 9 nm is 254 nm 2 .
  • a sum of the surface areas of 64 CDT multimers is 1.6 ⁇ 10 4 nm 2 . That is, the surface area per 100 nm of the length in the lengthwise direction of the titanium nano-rod structure encapsulating the CDT multimers observed in this case is 2.6 ⁇ 10 4 nm 2 , and could be estimated to be 2.4 times larger than 1.1 ⁇ 10 4 nm 2 that was the surface area per 100 nm of the length in the lengthwise direction of the titanium nano-rod structure which did not encapsulate the CDT multimer.
  • the iron oxide nanoparticle encapsulated in the CDT multimer could be introduced into the titanium film, it is expected to introduce the metal nanoparticle of nickel, cobalt, manganese, phosphorus, uranium, beryllium, aluminium, cadmium sulfide, cadmium selenide, palladium, chromium, copper, silver, gadolium complex, platinum cobalt, silicon oxide, cobalt oxide, indium oxide, platinum, gold, gold sulfide, zinc selenide, and cadmium selenium, which are predicted to be able to encapsulated in the CDT multimer, into a titanium film and a titanium oxide film which coat CNT.
  • CDT carbon nanotube single walled, Sigma, 519308
  • CNT carbon nanotube single walled, Sigma, 519308
  • 50 mM potassium phosphate buffer pH 6.0
  • 40 mL of the obtained solution was sonicated (200 W, 25%) on ice using Digital Sonifier (Branson, USA) in cycles of sonicating the solution for one second and resting the sonication for three seconds, and the sonication treatment was continued for 5 minutes.
  • a thick element having a diameter of 10 mm was used for the sonication.
  • the complex was further washed by adding 40 mL of water and centrifuging it. Finally, 0.8 mL of water was added and the solution was transferred to a 1.5 mL of microtube. Subsequently, 200 ⁇ L of the obtained CNT/CDT/Ti solution was placed on a quartz board and heated at temperature ranging from 450° C. to 800° C. (at each temperature of 500° C., 600° C., 700° C. and 800° C.) for 30 minutes (temperature rising rate: 50° C./minute).
  • Black powder obtained by the burning at each temperature was stained with 3% PTA, and analyzed under TEM. Transmission electron microscopic images of the structures obtained by heating show the complex of CNT and CNHBP-Dps-TBP (CDT multimer) coated with titanium oxide.
  • the black powder obtained by burning at 450° C. was analyzed by X ray diffraction (XRD).
  • XRD X ray diffraction
  • Peaks specific to (101) and (200) phases of anatase type TiO 2 crystal could be observed.
  • the CNT/CDT/Ti complex obtained in Example 14 was used as a material for a photoelectric conversion layer of an photoelectric conversion element (dye-sensitized solar cell) to evaluate its effect on properties of the dye-sensitized solar cell.
  • the production of the dye-sensitized solar cell was carried out by modifying the protocols by Solaronix.
  • a CNT(SWNT)/CDT/Ti corresponding to 1 mL of a reaction solution was synthesized by the above method, washed with water and suspended in an ethanol solution.
  • the CNT(SWNT)/CDT/Ti complex was blended into a titanium oxide paste (Ti-Nanoxide D, Solaronix), and used as a material of a dye-sensitized solar cell.
  • a photoelectric conversion layer pieces of mending tape (3M Company, a thickness of about 100 ⁇ m) cut into 5 mm and doubly attached were attached to both ends of an FTO substrate (fluorine-doped tin oxide, Solaronix), respectively that was a transparent electrode substrate cut into 25 mm ⁇ 25 mm. An interval between the tape pieces was 10 mm.
  • the titanium oxide paste containing the CNT(SWNT)/CDT/Ti complex was placed between the tape pieces, extended flatly using a slide glass, and left stand at 30° C. for 30 minutes to dry the titanium oxide paste.
  • the substrate on which the titanium oxide paste had been placed was placed in a burning furnace and burned at 450° C. for 30 minutes. The temperature was raised at 90° C./minute. After the burning, the substrate was naturally cooled to 100° C. or below. 1 mL of 0.2 g/L ruthenium (Ru) dye-sensitizing solution (N719, dissolved in dry ethanol, Solaronix) was applied to the burned substrate, which was then left stand at room temperature for 24 hours.
  • the electrode substrate stained red by being left stand for 24 hours was washed with ethanol to remove the dye not adsorbed to the titanium oxide surface, dried using a dryer, and used as a photoelectrode.
  • Ru ruthenium
  • a dye-sensitized solar cell was made using a Pt electrode (opposite electrode) obtained by coating the surface of the fluorine-doped tin oxide (FTO) film with platinum (Pt) having a thickness of 50 nm, and the structure comprising the above photoelectrode.
  • Pt electrode opposite electrode
  • FTO fluorine-doped tin oxide
  • Pt platinum
  • a sealing sheet (SX1170-25, Solaronix) was used as a sealing material, and heating at 120° C. for 5 minutes was given using a hotplate.
  • Araldite Rapid Showa Highpolymer Co., Ltd.
  • an epoxy-based adhesive was applied to an adhered surface not completely sealed and left stand at 30° C. for 2 hours to seal completely.
  • an iodine electrolyte solution (Solaronix) was added to obtain the dye-sensitized solar cell.
  • the produced dye-sensitized solar cell was evaluated by illuminating with light at an intensity of 100 mw/cm 2 with a xenon lamp.
  • An open voltage Voc (V), a short-circuit current density Jsc (mA/cm 2 ), a fill factor FF, and a photoelectric conversion efficiency ⁇ (%) were evaluated as properties of the dye-sensitized solar cell.
  • a rate of one converted into electric power in incident energy by the illuminated light is referred to as the photoelectric conversion efficiency ⁇ .
  • a current density measured when the voltage is 0 V is referred to as the short-circuit current density Jsc, and the voltage when a current does not flow is referred to as the open voltage Voc.
  • the short-circuit current density was 12 mA/cm 2 in the dye-sensitized solar cell [device 2 ( ⁇ )] comprising a photoelectrode formed of the titanium oxide paste alone.
  • the short-circuit current density was 15 mA/cm 2 in the dye-sensitized solar cell [device 1 (+)] using the titanium oxide paste in which the CNT (SWNT)/CDT/Ti complex had been blended as the functional material of the photoelectrode.
  • an amount of the current was increased by 25% by using the CNT (SWNT)/CDT/Ti complex as the functional material of the electrode.
  • the photoelectric conversion efficiency was increased to 1.4 times by using the titanium oxide paste in which the CNT (SWNT)/CDT/Ti complex had been blended as the functional material of the photoelectrode.
  • the CNT/CDT/Ti complex obtained in Example 14 was used as the material for the photoelectric conversion layer of the photoelectric conversion element (dye-sensitized solar cell), and effects of the complex on the properties of the dye-sensitized solar cell was evaluated.
  • the production of the dye-sensitized solar cell was carried out by modifying the protocol by Solaronix.
  • the CNT(SWNT)/CDT/Ti corresponding to 1 mL of a reaction solution was synthesized by the above method, washed with water and suspended in an ethanol solution.
  • the CNT(SWNT)/CDT/Ti complex was blended into the titanium oxide paste (Ti-Nanoxide D, Solaronix) so that the complex was contained at a final concentration of 0.2% by weight in a titanium oxide electrode after the burning, and used as the material of the dye-sensitized solar cell.
  • the FTO substrate fluorine-doped tin oxide, Solaronix
  • the transparent electrode substrate cut into 25 cm ⁇ 25 cm was immersed in an aqueous solution of 40 mM titanium tetrachloride at 80° C. for 30 minutes.
  • Tape pieces of the mending tape (3M Company, thickness: about 100 ⁇ m) cut into 5 mm and doubly attached were attached to the both ends of the FTO substrate. The interval between the tape pieces was 5 mm.
  • the titanium oxide paste containing the CNT(SWNT)/CDT/Ti complex was placed between the tape pieces, extended flatly using the slide glass, and left stand at 30° C. for 30 minutes to dry the titanium oxide paste.
  • the substrate on which the titanium oxide paste had been placed was placed in the burning furnace and burned at 450° C. for 30 minutes. The temperature was raised at 90° C./minute. After the burning, the substrate was naturally cooled to 100° C. or below.
  • a titanium oxide portion on the FTO substrate was cut into a 5 mm ⁇ 10 mm square, this substrate was immersed in 1 mL of 0.2 g/L ruthenium (Ru) dye-sensitizing solution (N719, dissolved in dry ethanol, Solaronix), and left stand at room temperature for 24 hours.
  • the electrode substrate stained red by being left stand for 24 hours was washed with ethanol to remove the dye not adsorbed to the titanium oxide surface, dried at room temperature, and used as a photoelectrode (device 11).
  • a device using a photoelectrode made from a titanium oxide electrode containing 0.2% by weight of a CNT/titanium oxide complex synthesized by oxidation treatment (device 12), a device using a photoelectrode made from a titanium oxide electrode containing 0.2% by weight of CNT (device 13), and a device using a photoelectrode made from a titanium oxide electrode containing no CNT (device 14) were made.
  • the CNT/titanium oxide complex was synthesized by the oxidation treatment with reference to the method in the reference (W. Wang et al. (2005) Journal of Molecular Catalysis A: Chemical, 235, 194-199).
  • a dye-sensitized solar cell was made using a Pt electrode (opposite electrode) obtained by coating the surface of the fluorine-doped tin oxide (FTO) film with platinum (Pt) having a thickness of 50 nm, and the structure comprising the above photoelectrode.
  • Pt electrode opposite electrode
  • FTO fluorine-doped tin oxide
  • Pt platinum
  • a sealing sheet (SX1170-25, Solaronix) was used as a sealing material, and the heating at 120° C. for 5 minutes was given using the hotplate.
  • Araldite Rapid Showa Highpolymer Co., Ltd.
  • the epoxy-based adhesive was applied to the adhered surface not completely sealed and left stand at 30° C. for 2 hours to seal completely.
  • the iodine electrolyte solution (Solaronix) was added to obtain the dye-sensitized solar cell.
  • the produced dye-sensitized solar cell was evaluated by illuminating with light at an intensity of 100 mW/cm 2 with the xenon lamp.
  • the larger short-circuit current density was measured in the dye-sensitized solar cells comprising the photoelectrode formed from the titanium oxide paste containing a nanomaterial (devices 11, 12, and 13) than in the dye-sensitized solar cell comprising the photoelectrode formed from the titanium oxide paste alone (device 14).
  • the short-circuit current density was increased by 180% by using the CNT(SWNT)/CDT/Ti complex as the functional material of the electrode.
  • the short-circuit current density in the device using the complex of titanium oxide and CNT synthesized using CDT (device 11) as the functional material of the electrode was larger than the short-circuit current density in the device using CNT alone as the functional material of the electrode (device 13) and the short-circuit current density in the device using the complex of titanium oxide and CNT synthesized by the oxidation treatment as the functional material of the electrode (device 12).
  • the photoelectric conversion efficiency ⁇ in the device using the titanium oxide paste in which CNT(SWNT)/CDT/Ti had been blended (device 11) as the functional material of the photoelectrode was 2.2 times larger than the photoelectric conversion efficiency ⁇ in the device using the titanium oxide paste not containing CNT (device 14) as the functional material of the photoelectrode.
  • the highest photoelectric conversion efficiency was measured in the device using the titanium oxide paste in which CNT(SWNT)/CDT/Ti had been blended as the functional material of the photoelectrode among the devices produced this time. Therefore, it was found that the performance of the solar cell could be enhanced by using the complex of titanium oxide and CNT synthesized using CDT under the mild condition.
  • the metal-encapsulating protein Dps from Listeria innocua the N-terminus of which is fused with the carbon nanohorn-binding peptide (abbreviated as CNHBP and consisting of the amino acid sequence DYFSSPYYEQLF (SEQ ID NO:6), see International Publication No. WO2006/068250), and the C-terminus of which is fused with a zinc oxide-precipitating peptide (abbreviated as ZnO1′ and consisting of the amino acid sequence EAHVMHKVAPRPGGGSC (SEQ ID NO:30), see Umetsu et al., Adv. Mater., 17, 2571-2575 (2005)) was constructed (abbreviated as CNHBP-Dps-ZnO1′ or CDZ, SEQ ID NOS:31 and 32) by the following procedure.
  • CNHBP carbon nanohorn-binding peptide
  • ZnO1′ zinc oxide-precipitating peptide
  • EAHVMHKVAPRPGGGSC S
  • PCR was carried out using pET20-CDT as the template DNA, and the oligonucleotides consisting of the nucleotide sequence represented by SEQ ID NO:11 and the nucleotide sequence of tttGGATCCttaAcaACTAccTccAccAggAcGTggAgcAacTttAtgcatTacAtgTg cTtcttctaatggagctttttc (SEQ ID NO:33) as the primers.
  • SEQ ID NO:33 the PCR product was purified using Wizard SV Gel and PCR Clean-Up System (Promega, USA) and digested with the restriction enzymes DpnI and BamHI.
  • the PCR product digested with the restriction enzymes was self-ligated using the T4 ligase (Promega, UAS).
  • E. coli BL21 (DE3) (Nippon Gene, Japan) was transformed with the self-ligated PCR product to construct BL21 (DE3) possessing the expression plasmid carrying the gene encoding Dps (CDZ), the N-terminus and the C-terminus of which were fused to the carbon nanohorn-binding peptide and the zinc oxide-precipitating peptide, respectively (pET20-CDZ).
  • BL20 (DE3)/pET20-CDZ was inoculated to 100 mL of the LB medium (containing 100 mg/L of ampicillin), and cultured with shaking using a 500 mL flask at 37° C. for 24 hours.
  • the resulting microbial cells were collected by centrifugation (6,000 rpm, 5 minutes) and suspended in 5 mL of 50 mM Tris-HCl buffer (pH 8.0).
  • the microbial solution was sonicated to disrupt the microbial cells.
  • the resulting solution was centrifuged at 6,000 rpm for 15 minutes to collect a supernatant fraction.
  • the collected solution was heated at 60° C. for 20 minutes and then cooled on ice rapidly after the heating.
  • the cooled solution was centrifuged at 6,000 rpm for 15 minutes to collect a supernatant (about 5 mL) again.
  • This solution was sterilized using the disc filter (Millex GP 0.22 ⁇ m, Millipore, USA).
  • This solution was ultrafiltrated and concentrated using Amicon-Ultra-15 (NMWL. 50000, Millipore, USA), and the buffer in which the protein was dissolved was replaced with Tris-HCl buffer (50 mM Tris-HCl solution, pH 8.0) to obtain 2.5 mL of a protein solution.
  • Anion exchange chromatography was used for purifying CDZ from the resulting protein solution. Specifically, 2.5 mL of the protein solution was applied to HiLoard 26/10 Q-Sepharose High Performance column (GE healthcare, USA) equilibrated with 50 mM Tris-HCl buffer (pH 8.0). The separation/purification was carried out at a flow rate of 4.0 mL/minute by making the concentration gradient of the salt from 0 mM to 500 mM NaCl in 50 mM Tris-HCl buffer (pH 8.0), and fractions containing CDZ were collected. Further, the collected solution was ultrafiltrated and concentrated using Amicon-Ultra-15 (NMWL. 50000, Millipore, USA), and the buffer in which the protein was dissolved was replaced with pure water to obtain a CDZ solution.
  • Amicon-Ultra-15 NMWL. 50000, Millipore, USA
  • CDZ dissolved in 50 mM Tris-HCl buffer (pH 8.0) before the buffer was replaced with pure water was stained with 3% PTA (phosphotungstic acid), and analyzed under the transmission electron microscope. As a result, it was found that CDZ formed the cage-like multimer having a diameter of about 9 nm, as is similar to CDT.
  • CDZ, CDT, or CD dissolved in pure water was added at a final concentration of 0.1 mg/mL to an aqueous solution of 0.1 M zinc sulfate. After the solution was left stand at room temperature for one hour, a turbidity of the solution was measured using the light at 600 nm. A white precipitate occurred prominently in the solution containing CDZ. It was thought that this white precipitate was zinc hydroxide or zinc oxide. On the other hand, no precipitate occurred at all in the solution containing no protein. It is known that zinc hydroxide becomes zinc oxide by being heated at about 125° C. From above, it was suggested that CDZ had an activity to precipitate the zinc compound.
  • CDZ and CNT (Sigma, 519308, carbon nanotube single walled) were added at a final concentration of 0.3 mg/mL to potassium phosphate buffer (50 mM, pH 6.0).
  • the ultrasonic pulse 200 W, duty 206) for one second with an interval of 3 seconds was given to this solution for 5 minutes using Digital Sonifier 450 (Branson, USA).
  • the sonicated CDZ/CNT mixed solution was centrifuged (15,000 rpm, 5 minutes), a protein/CNT complex included in a supernatant was stained with 3% PTA and observed under the transmission electron microscope (JEM-2200FS, 200 kV).

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